汉防己甲素衍生物HL-49对Bloom DNA解旋酶生物学特性的影响

Effects of tetrandrine derivative HL-49 on biological property of Bloom DNA helicase

目的汉防己甲素衍生物是一类双苄基异喹啉类化合物,具有明显的抗肿瘤活性。研究汉防己甲素衍生物HL-49对Bloom DNA解旋酶(bloom DNA helicase,BLM)构象和生物学活性的影响对于探索其抗肿瘤作用机制具有重要意义。方法采用紫外光谱扫描研究药物HL-49对BLM DNA解旋酶构象的影响,荧光偏振技术和孔雀绿-磷钼酸铵比色法分析HL-49对BLM解旋酶的DNA结合活性、DNA解链活性、ATPase活性。结果紫外光谱扫描结果显示,HL-49对BLM DNA解旋酶的三维构象影响不明显。荧光偏振法结果显示,HL-49对BLM解旋酶的DNA(dsDNA/ssDNA)结合活性的影响不明显,但随浓度的增加,与DNA相互结合的能力越强(P<0.01)。随着HL-49浓度的增加,BLM DNA解旋酶的DNA解链能力下降,K_(obs)值逐渐降低。孔雀绿-磷钼酸铵比色结果说明,随着HL-49浓度的增加或作用时间延长,HL-49能明显抑制BLM DNA解旋酶的ATPase活性。结论汉防己甲素衍生物HL-49通过与DNA的可逆性结合,抑制BLM DNA解旋酶的ATPase活性和DNA解链活性。

Aim To study the effect of tetrandrine derivative HL-49 on the conformation and biological activity of Bloom helicase(BLM),and to explore its anti-tumor mechanism.Methods The effect of HL-49 on the conformation of BLM helicase was studied by ultraviolet spectroscopy.The effects of HL-49 on DNA binding activity,DNA chain dissociation activity and ATPase activity of HL-49 on BLM DNA helicase were analyzed by fluorescence polarization and malachite green-ammonium phosphomolybdate colorimetric method.Results HL-49,a tetrandrine derivative,indirectly inhibited the ATPase activity of BLM DNA helicase and DNA unwinding activity by reversible binding with DNA.The results of fluorescence polarization experiments showed that HL-49 could not affect the binding activity of BLM DNA helicase to DNA(dsDNA/ssDNA),but could bind to DNA in a concentration-dependent manner(P<0.01).With the increase of HL-49 concentration,the DNA unwinding ability of BLM DNA helicase decreased,and the K_(obs) value decreased gradually.The results of malachite green-ammonium phosphomolybdate colorimetry showed that HL-49 could significantly inhibit the ATPase activity of BLM DNA helicase.Conclusions HL-49 can inhibit the ATPase activity and DNA unwinding activity of BLM DNA helicase by the reversible binding with DNA.